© 2018 by the authors. Organic matter (OM) has an essential biogeochemical influence along the hydrological continuum and within aquatic ecosystems. Organic matter derived via microbial processes was investigated within a range of model freshwater samples over a 10-day period. For this, excitation-emission matrix (EEM) fluorescence spectroscopy in combination with parallel factor (PARAFAC) analysis was employed. This research shows the origin and processing of both protein-like and humic-like fluorescence within environmental and synthetic samples over the sampling period. The microbial origin of Peak T fluorescence is demonstrated within both synthetic samples and in environmental samples. Using a range of incubation temperatures provides evidence for the microbial metabolic origin of Peak T fluorescence. From temporally resolved experiments, evidence is provided that Peak T fluorescence is an indication of metabolic activity at the microbial community level and not a proxy for bacterial enumeration. This data also reveals that humic-like fluorescence can be microbially derived in situ and is not solely of terrestrial origin, likely to result from the upregulation of cellular processes prior to cell multiplication. This work provides evidence that freshwater microbes can engineer fluorescent OM, demonstrating that microbial communities not only process, but also transform, fluorescent organic matter.